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Diabetes Approach by Multi-Organ-on-a-Chip

Periodic Reporting for period 4 - DAMOC (Diabetes Approach by Multi-Organ-on-a-Chip)

Reporting period: 2021-07-01 to 2021-12-31

Diabetes is the most prevalent and epidemic metabolic disorder throughout the world. Type 2 diabetes (T2D) is the most common form characterized by hyperinsulinemia and insulin resistance. The maintenance of normal glucose homeostasis depends on a finely balanced dynamic interaction between skeletal muscle sensitivity to insulin and insulin secretion. Thus, the evolution of T2D requires the presence of defects in both insulin secretion and insulin action, and it requires the simultaneous study of both tissues, which results in complex, expensive and time consuming with conventional methods.

The pharmaceutical industry relies heavily on in vivo animal models and in vitro two-dimensional (2D) cell cultures to develop therapeutic strategies. There are many ethical issues surrounding animal studies, and serious concerns also exist regarding their biological relevance to humans. Current in vitro tissues are also helpful for studying the molecular and cellular basis of physiological and pathological responses of biological processes. However, due to their 2D structure, they do not consider the complexity of the physiological microenvironment in which cells grow. There is, thus, growing interest in developing fully functional three-dimensional (3D) tissues.
DAMOC aims to overcome these limitations in a revolutionary technological approach that allows us to engineer skeletal muscle tissues and pancreatic islets in a multi-OOc to open new research areas on human T2D disease.

During DAMOC's execution period, we have accomplished all objectives. We have bioengineered a new in vitro model to mimic the insulin-mediated skeletal muscle glucose metabolism. To this aim, both muscle tissues and pancreatic islets have been fabricated, generated and combined in a multi-OOC approach to study pancreatic islets' insulin secretion and the associated glucose-induced contraction of muscle tissues. We have developed several biomaterials and protocols to encapsulate and maturate skeletal muscle cells and pancreatic pseudoislets. Engineered tissues have benefited from novel scaffolds and have been integrated into bioreactors, with an electrical stimulator and biosensors to monitor myokine secretion from skeletal muscle cells, insulin production, and effects of skeletal muscle contraction on beta-cells. In a multidisciplinary approach, we have used micro- and nanoscale fabrication technologies developed by our research group, and we have integrated novel biosensing technology to monitor metabolic processes relevant to diabetes, such as myokines release. This multi-OOC is an important enabling step for diabetes modelling, the study of insulin resistance, and the investigation of drug candidates for therapy, usually performed by long-time and expensive animal experiments.
Skeletal muscle: We have studied three different composite biomaterials as bioinks to produce 3D engineered skeletal muscle myotubes. We have demonstrated that several composite materials allow the bioprinting of 3D constructs of skeletal muscle that can be used in in vitro applications promoting their functionality and preserving their structure. In this study, it is vital to match not only the morphology of the functional skeletal muscle fibers, but also the cellular arrangement, controlling hydrogel properties, which are critical for proper cellular function and tissue morphogenesis.

Beta-cells: We worked in the production of 3D beta-cells aggregates with controllable sizes in hydrogels using 3D bioprinter technology. We reproduced the 3D architecture of pancreatic islets through 3D bioprinting in 3D spherical structures. Insulin release has been measured to determine encapsulated cell aggregates' functionality and maturation.
The capacity to secrete insulin by the pseudoislets was evaluated by a glucose-stimulated insulin secretion assay. We showed that encapsulated pancreatic cells maintain their β-cell identity and their capacity to secrete insulin.

Biosensors and organs on a chip: Two different types of biosensors have been developed and tested during the project, electrochemical and plasmonic biosensors. For electrochemical biosensors, we used screen-printed gold electrodes (SPGEs), where we previously immobilized antibodies against IL-6 and TNF-α, binding the cytokines present in the medium. Plasmonic biosensors were fabricated by a simple and reproducible fabrication process using nanostructures as polymeric templates containing nanogratings.

Multi-OOC integration: Multi-OOC experiment was set up integrating the Multi-OOC with the PBP to monitor in real-time the cross-talk effect of the IL-6 secreted by an electrically stimulated skeletal muscle tissue over the glucose secretion of the pseudoislets, (simulating the native pancreas model).
The upcoming engineered microtissues represent a new paradigm in the field of in vitro assays, providing in vitro systems with tissue-like in vivo functionality. Engineered microtissues are available because of the combination of microfabrication, microfluidics, tissue-engineering components, biosensors, and specific functional cells.
We have screened and optimized the physical properties of different composite hydrogels for the growth and development of muscle fibers. They have shown good biocompatibility and bioactivity, and, in contrast with other hydrogels, they are long-lasting materials. Thus, they are good candidates as biomaterials for in vitro applications and as bio-actuators. These hydrogels have the potential to meet an assortment of cellular and mechanical demands required for engineering tissues. The combination of hydrogel composites with bioprinting methods allowed us to efficiently obtain 3D structures of differentiated and aligned muscle fibers, which is highly interested in skeletal muscle tissue engineering. We obtained pancreatic cells of the controllable size using a similar composite biomaterial and 3D bioprinting technology. The proposed technique can be used in cell therapy and tissue regeneration applications.

We have identified several results of the project that can be subject to intellectual protection or that offer a potential for future technology transfer and exploitation. Among them, 3D engineered microtissues. More specifically, the protocol of fabrication of pancreatic islets and skeletal muscle fibers by 3D bioprinting technology (all biomaterials and bioinks used in the protocol included) with controllable size and maintaining a long-lasting functionality could attract not only in vitro applications but also in vivo transplants for patients with type I diabetes.

In addition, we have explored the development of optical biosensors in the last years, especially those based on metal nanostructures. Plasmonic biosensors based on metallic nanostructures have a high potential for sensitive, label-free, multiplexed, and real-time biodetection in integrated lab-on-a-chip platforms.
Furthermore, nanoplasmonic sensors have been developed based on metallic nanogratings using Blu-Ray optical discs as low-cost polymer disposable nanostructured templates.This nanoplasmonic biosensor recently allowed the direct and label-free monitoring of albumin over time in a 2D fatty liver disease model under flow conditions using highly specific polyclonal antibody with a LOD in the pM order without any amplification or pretreatment of the sample.
Pseudoislets generated in CMC-based cryogel scaffolds to study pancreatic islet function
IL-6 and insulin are secreted as the muscle contraction response induced by EPS.
Design and fabrication of the microfluidic Multi-OOC device
IL-6 and Insulin plasmonic biosensor
Bioengineered skeletal muscle respond to electric pulse stimulation